Cosmetic compositions containing microcrystalline collagen,a water-insoluble,ionizable,partial salt of collagen

ABSTRACT

1. A COMPOSITION OF MATTER IN THE FORM OF A COSMETIC CREAM OR LOTION COMPRISING AMIXTURE OF AT LEAST ONE CONVENTIONAL COSMETIC CREAM OR LOTION INGREDIENT OTHER THAN WATER AND A STABLE DISPERSION IN WATER OF A WATER INSOLUBLE, IONIZABLE PARTIAL SALT OF COLLAGEN HAING A BOUND IONIZABLE ACID CONTENT OF FROM ABOUT 0.4 TO HAVING A 0.7 MILLIMOLE OF ACID (CALCULATED AS HCL) PER GRAM OF COLLAGEN BASED COLLAGEN CONTAINING APPROXIMATELY 0.78 MILLIMOLE UPON COLLAGEN CONTAINING APPROXIMATELY LAGEN, BEING ESSENTIALLY FREE OF TROPOCOLLAGEN AND DEGRADED DERIVATIVES THEREOF AND BEING FURTHER CHARACTERIZED IN THAT WHEN COLLODIALLY DISPERSED IN WATER TO FORM A 1/2% BY WEIGHT DISPERSION WHEREIN AT LEAST 10% BY WEIGHT OF THE PARTIAL SALT HAS A PARTICLE SIZE NOT EXCEEDING 1 MICRON, THE DISPERSION EXHIBITS A PH OF ABOUT 3.2+0.2 AND EXHIBITS AN ESSENTIALLY CONSTANT VISCOSITY AFTER ABOUT 1 HOUR OF FOR AT LEAST 100 HOURS WHEN STORED IN A CLOSED CONTAINER AT 5*C., THE COSMETIC INGREDIENT BEING COMPATIBLE WITH THE PARTIAL SALT OF COLLAGEN, BEING PRESENT IN AN AVAILABLE FORM AND IN AN AMOUNT SUFFICIENT TO IMPART ITS CHARACTERISTIC COSMETIC PROPERTY TO THE COMPOSITION.

United States Patent O ce COSMETIC COMPOSITIONS CONTAINING MICRO-CRYSTALLINE COLLAGEN, A WATER-INSOLU- BLE, IONIZABLE, PARTIAL SALT OFCOLLAGEN Orlando A. Battista, Fort Worth, Tex., assiguor to Avicon,Inc., Fort Worth, Tex.

No Drawing. Application Feb. 2, 1970, Ser. No. 12,504, now Patent No.3,691,281, dated Sept. 12, 1972, which is a division of abandonedapplication Ser. No. 586,969, Oct. 17, 1966, and a continuation ofapplication Ser. No. 14,709, Feb. 9, 1970, now Patent No. 3,628,974;said application Ser. No. 586,969 being a continuationin-part ofabandoned application Ser. No. 436,371, Mar. 1, 1965. Divided and thisapplication June 19, 1972, Ser. No. 264,176

Int. Cl. A61k 7/00, 7/06, 7/16 U.S. Cl. 424-359 8 Claims ABSTRACT OF THEDISCLOSURE Cosmetic compositions which include in addition to thecosmetic ingredient or ingredients a water-insoluble, ionizable, partialsalt of collagen or microcrystalline collagen. The composition may be inthe form of a pourable liquid, a gel or ointment or a solid compressedtablet. The microcrystalline collagen colloidally dispersed in theliquid improves the stability of suspended ingredients in liquids andgels.

This application is a division of application Ser. No. 12,504, filedFeb. 2, 1970, now Pat. No. 3,691,281, dated Sept. 12, 1972, which is adivision of application Ser. No. 586,969, filed Oct. 17, 1966, abandonedin favor of continuing application Ser. No. 14,709, filed Feb. 9, 1970,now Pat. No. 3,628,974, dated Dec. 21, 1971, application Ser. No.586,969 being a continuation-in-part of application Ser. No. 436,371,filed Mar. 1, 1965, now abandoned.

This invention relates to a new form of collagen which has markedutility compared with present known forms of collagen, and to foodproducts, cosmetic compositions and pharmaceutical preparationscontaining the new form of collagen. This new water-insoluble,microcrystalline colloidal form of collagen, because it iswater-insoluble and insoluble in aqueous solutions having a pH as low as3-4, is capable of producing aqueous gels made from collagenderivatives, and produces superior end products.

Collagen is the principal building block of the hides and skins of mostmammals, including man, and its principal source is hide substance. Italso is the chief constituent of many other parts of mammals, such astendons, intestinal walls, etc. In addition to its principal usein themanufacture of leather-collagen is also widely used in the preparationof such materials as glue and gelatin. More recently, much study hasbeen directed to the solubilization of collagen, and the reconstitutionof the solubilized collagen as fibers for use as sutures, and as fibrousmats for various purposes.

The elementary basic molecular unit of collagen is tropocollagen,sometimes called procollagen. This unit has been isolated, and electronmicrographs made, so that its structure is well understood. Themacromolecules consist of three polypeptide chains coiled together in along helix, about 10 to A. units in diameter and about 3000 A. units(0.3 micron) long. Tropocollagen is insoluble in neutral water, but issoluble in certain salt solutions and in dilute acid solutions having apH of about 3 and lower. Much of the work done in the production ofreconstituted collagen products has involved the conversion of fibrouscollagen to acid-soluble tropocollagen usually employing. relativelysevere acid pretreatments whereby the collagen fibers and fibrils arereduced to tropocollagen molecules as above described 3,839,590 PatentedOct. 1, 1974 followed by reprecipitation of the molecularly dispersedtropocollagen into a reconstituted form. A typical example of thisprocedure is disclosed in US. Pat. 3,157,524.

The next higher organized state in which collagen has heretofore beenknown is the collagen fibril, which consists of long, thin strandscomprising thousands of individual tropocollagen units; the fibrils maybe several hundred to a thousand angstrom units in diameter, and vary inlength, generally being tens of microns long. In this form, the collagenis initally water-insoluble, not only at the neutral point but also inacidified water having a pH of about 3 and lower. It is these fibrilswhich associate to form the macroscopic fibers present in naturalsubstances and which fibers comprise many thousands of fibrils bondedtogether.

The art has long worked with these collagen fibers with the thought ofusing them for the formation of water-absorbent mats or sponges whichcould be used for various purposes. Originally, what was attempted wasthe breaking down of the fibers of the hide into their individualmolecular units (tropocollagen and/or gelatin) by solubilizing them andthen reconstituting them into batts. This process is extremely difiicultand expensive.

The second type of approach is disclosed in US. Pat. No. 3,157,524. Thispatent discloses that batts or sponges may be formed by freezing andacidified collagen gel comprising substantial amounts of tropocollagen,after which the water is sublimed under high vacuum while maintainingthe temperature below the freezing point. The patent points out thatsuch products redissolve in water and attempts to neutralize the acid inthe frozen product by aqueous alkaline solutions destroys the desiredfoam-like texture and produces a mat structure that loses much of itsutility. The patentee overcomes this difficulty by freezing a gel ofwater-soluble, acid treated collagen fibers, immersing the frozen massin a circulating bath of a water-miscible solvent containing an alkalineagent to neutralize the acid whereby the collagen fibrils are dehydratedand coagulated and the salt formed by the neutralization is removed, andsubsequently again drying the resultant collagen mass to form asponge-like mat which will resist dissolution in water.

The use of this method for neutralization is both slow and costly, andinvolves several processing steps which are expensive. Moreover, thereis some loss of porosity in the sponges due to collapse in the organicsolvent. Most importantly, the reconstituted collagen has lost itsoriginal morphology, and the natural bonds between the tropocollagenunits present in the original fibrils are substantially weakened by thesolubilization, regeneration and neutralization steps used.

The present invention provides a new form of collagen, which is adistinctly new physical state intermediate between that of swollencollagen fibers and the tropocollagen molecules disclosed in the abovepatent. This new physical form of collagen is microcrystalline andcolloidal; it consists of bundles of aggregated tropocollagen unitswhich vary in length up to just under one micron, and in diameter fromabout twenty-five angstrom units to some hundreds of angstrom units.Compositions comprising various forms of collagen, at least about tenpercent by weight of which comprises my new submicron microcrystallinecolloidal collagen particles, and which are substantially free oftropocollagen and degraded derivatives thereof, produce viscosity-stableaqueous gels at low concentrations, of the order of one-half percent.This is possible because of the strict control of acid concentration incombination with appropriate mechanical disintegration to produce themicrocrystalline, water-insoluble particles and to preclude truesolution.

This is in sharp contrast to the type of aqueous gel formed bytropocollagen, and degraded forms of collagen such as gelatin and insharp contrast to products made in accordance with the above patent,where the collagen has been solubilized and reprecipitated. The gelsformed by the water-soluble and/or dilute acid-soluble forms of collagenthicken on standing to produce rubbery masses, so that their utility issharply curtailed by this lack of stability. Moreover, gels may beproduced in accordance with this invention utilizing much lowerpercentages of solids. At this stage, the particles are completelyundenatured, there has been a minimal disruption of the original lateralbonding forces between the tropocollagen units comprising the originalfibrils and many of the original lateral natural bonding forces remainsubstantial- 1y unchanged.

The water-insoluble microcrystalline colloidal collagen is prepared fromany undenatured collagen in the natural state, either as pieces oforiginal hide, gut, or other high-collagen source, but preferably withpieces dried under non-denaturing conditions and chopped up for easierhandling. The undenatured collagen is treated very carefully undercontrolled conditions with very dilute acid solutions the pH of which isfrom about 1.6 to 2.6. Where the source material is wet, the proportionof water present must be taken into consideration in preparing the acidsolution to be used in the treatment of the source material. Thematerial is then mechanically disintegrated, in the presence of thedilute acid, until about ten percent or more of the material is reducedto submicron size. It is not essential that all the source material bereduced to submicron size. The product becomes useful when about tenpercent has been so reduced, although optimum results are obtained atsubstantially higher concentrations of the submicron microcrystallinematerial.

Alternatively, the acid treated, undenatured collagen may be driedwithout disintegration. Subsequently, in the production of variousproducts, for example, water base paints, the dried, acid treatedcollagen is subjected to disintegration during conventional mixingoperations.

In producing my product, it is essential that the starting material beundenatured. Satisfactory raw materials from which the products of thisinvention have been prepared include fresh cowhides and calfhides,salted down cowhides, wet moosehide, and sun-dried pigskins, sheepskinsand goatskins such as are conventionally used for making leather, aswell as a special technical hide collagen prepared from hide splits andpossessing a minimal reduced bacterial count (circa 200-4000 bacteriaper square inch). Similarly, hides and skins which have been freezedried or dried by solvent displacement methods to maintain the collagenin a substantially undenatured state are equally satisfactory. Ingeneral, if a hide is in such condition that it can be Wet back for usein tanning to produce satisfactory leather, it is satisfactory forproducing microcrystalline colloidal collagen and the attendant stabledispersions. The preferred raw materials, because of its wideavailability and low cost, is obviously cowhide or technical grades ofcollagen prepared from cowhide and other animal hides.

In preparing the crude cowhides for use herein, it is obviouslydesirable to remove hair and flesh, so that they will not introducedimpurities which must later be removed. I find it advantageous to usedegrained hide especially when refined stable dispersions are desiredsince degraining removes some tightly bound impurities.

When operating next to an abbatoir, the hides may be processed fresh.However, it is desirable to use dry hides, for easier control ofprocessing. Solvent extraction of the water in known fashion may bepracticed; it is preferable to freeze dry the wet hides or finelycomminuted wet hides, and sublime the water off under vacuum. Thistechnique has marked advantages in insuring uniformity of raw materialas well as convenience and stability in storing and handling.

Undried split hides may also be stored at room temperature in a mixtureof water and a water-miscible organic solvent, to preserve them frombacterial attack during storage and handling at room temperature. Forexample, a 25/75 ethyl alcohol/water medium or a 25/75 isopropylalcohol/water medium may be used for such preservation of the hides atroom temperature, depending on whether or not they are to be convertedfor internal or topical applications, respectively.

The hide substance is preferably chopped up before treatment for ease ofhandling. Preferably, it is ground to reasonable fineness, for example,in an Urschel mill, to insure more uniform reaction, and to reduce thesubsequent energy required in the ultimate comminution to submicronsize. It should be noted that stable water-insoluble microcrystallinecolloidal dispersions of untreated hide substance containing therequired minimum amount of colloidal microcrystalline collagen particlescannot be obtained without the appropriate chemical pretreatmentemploying carefully controlled acid conditions in combination with theappropriate amount of mechanical disintegration.

For making microcrystalline colloidal collagen, an essentialpretreatment is a thorough soaking of the hide substance with verydilute acid at the required pH. The concentration needed with anyparticular acid varies with its composition and degree of dissociation;the resultant products are more or less useful, depending on thesefactors.

With hydrochloric acid and a typical vacuum freezedried cowhide, it isessential that the pH of the treating solution not exceed about 2.6 toproduce the microcrystalline colloidal collagen upon subsequentdisintegration. Optimum results are attained with acid solutions havinga pH of the order of 2 at 1% solids. Treatment with solutions having apH of less than about 1.6 causes rapid degradation of molecular weightwith an attendant buildup of acid-soluble tropocollagen and otherdegradation products as evidenced by a marked drop in apparent viscosityof the dispersed material upon disintegration in the aqueous liquid. Theoptimum pH of the treating solution will vary slightly depending uponthe specific portion of the animal hide and the age of the animal fromwhich the hide is obtained. In general, the optimum pH of the acidsolution varies indirectly with the age of the animal; that is, forhides from older animals, the optimum pH of the acid solution ispreferably in the lower portion of the pH range.

The action of the acid is three-fold. First, the acid serves to cause alimited swelling of the fibers. Second, there is a limited hydrolysis ofselective peptide linkages within the non-crystalline or amorphousregions of the collagen fibrils so that subsequent mechanicaldisintegration permits a ready fragmentation of the weakened morphologyinto microcrystalline particles having dimensions intermediate betweenthose of tropocollagen and collagen fibrils. Third, a portion of theacid reacts with available primary amino groups of the collagen to formwhat may be termed a collagen hydrochloride salt which, of course, isionized in the presence of water.

When the proportion of collagen source material is increased, the pH ofthe initial treating solution must be decreased because of the removalof a portion of the acid by reaction with the collagen. Thus, at aconcentration of 7.5% collagen, for example, it is necessary to utilizea hydrochloric acid solution having an initial pH of about 1.52. On theother hand, if the pH of the solution has. an initial pH of about 1.4,the collagen will be degraded excessively which is evidenced by aradical decrease in viscosity of the dispersion formed upon mechanicaldisintegration of the treated product.

After the acid treatment, the hide substance, with the acid distributedtherethrough, is subjected in mechanical attrition in the presence ofthe aqueous liquid to reduce at least about ten percent of the productto submicron size. In general, the preferred disintegrating equipmentsubjects the particles of treated collagen to high shear against eachother, such as the Waring Blendor and the Cowles Dissolver for lowsolids concentrations, causing disruption and effective reduction insize of the sub-fibril microcrystalline aggregates. High shear can beimparted in other ways, as by extrusion through small orifices as by theuse of a Rietz Extructor or by high pressure filtration through asintered plate particularly in the case of high (above 5%) solidsconcentrations, or other known techniques.

Preferably, the distintegration is continued well beyond the point whereten percent of the product is submicron, preferably until 25% to 85% ormore of the product has been reduced to colloidal size.

Hydrochloric acid has been referred to in the foregoing description andis also used in the examples merely because it is relatively inexpensiveand allows ready flexibility and ease of control. Other acids, bothinorganic and ionizable organic acids, such as, for example, sulfuricacid, hydrobromic acid, phosphoric acid, cyanoacetic acid, acetic acid,citric acid and lactic acid are satisfactory. Sulfuric acid, forexample, is satisfactory, but control of the action is difiicult. Citricacid may be substituted for hydrochloric acid with about equal results.Ease of control has reference to the ability to arrest the swelling andhydrolysis of the collagen fibers at that point whereby the insolublecolloidal material is formed and is retained while preventing the rapiddegradation of the material to a water-soluble product.

Alternatively, the ground hide substance after treatment with therequired acid solution may be dried under conditions that maintain thecollagen in a substantially undenatured state and stored or shipped inthe dried state. As a further alternative, the ground hide may beintimately mixed with a pulverized acid such as citric acid or tartaricacid. Prior to use in the production of a desired product, the drymaterial is mixed with the required amount of water, subjected tomechanical disintegration and then mixed with the other ingredients ofthe end product. Alternatively, the dry material is mixed with otheringredients of a desired product and with water and the mixture thensubjected to a mechanical attrition step wherein the hide substance isattrited and all ingredients become intimately mixed. For example, inthe use of the water-insoluble microcrystalline colloid substance as abinder in water-laid webs, the dry material may be mixed with water andmechanically disintegrated in a Cowles Dissolver and the resultingdispersion added to a pulp slurry before the sheeting operation.Alternatively, the dry material may be added to a pulp slurry and themixture then subjected to mechanical disintegration, as in a BauerRefiner, wherein the collagenous material is reduced to the requiredcolloidal size before the sheeting operation.

Upon completion of the disintegration, the gels produced have a pH offrom about 2.6 to 3.8, the specific pH being dependent upon the pH ofthe treating acid. It is essential that whatever the concentration ofthe collagen source material, the initial pH of the treating acidsolution be such that upon mechanical disintegration of the treatedcollagen, the pH of the solution be within the range of from about 1.6to about 2.6. The pH of the gels exhibiting optimum properties isbetween 3.0 and 3.4. For example, in the preparation of 1% gel, one partof finely ground, vacuum freeze-dried cowhide was treated with 100 partsof a hydrochloric acid solution having a pH of 2.25. After a minutetreatment in a Waring Blendor, the gel had a pH of 3.25. A 2% gel wasprepared in like manner and had a pH of 3.3. When one gram samples ofmats prepared by freeze drying these gels were placed in 100 mls. ofdistilled water, the partial hydrochloride salt of collagen ionizedwithout a disintegration of the mats and the pH of the water was loweredto a pH of 3.1.

For many uses, it is highly desirable to remove as much of the freefatty material present in the microcrystalline collagen aciddispersions. This removal may'be achieved by adding cellulosic fibers inthe form of highly bleached kraft wood pulp or microcrystallinecolloidal cellulose to the dispersion with appropriate mixing todistribute uniformly the cellulosic material throughout the dispersion.Subsequent filtration of the dispersions, as by a conventional pressurefiltration method utilizing layers of cellulosic fabric, cotton battingand the like mounted between suitable foraminous metal plates, resultsin a significant removal of the natural fatty materials present in theraw material. Alternative procedures to reduce such fatty materials tominimal levels are to extract the raw undried hides with organic liquidssuch as acetone, that Will dissolve fatty materials, or to force thedispersions through cellulose paper or fabric filters under very highpressures. Such filtration steps furthermore help to remove extraneoussmall amounts of other impurities such as chips of hair and fleshytissues that are quite undesirable in the finished products.

The invention may be illustrated by the examples which follow:

EXAMPLE 1 Ground, vacuum freeze dried cowhide was soaked in an aqueoushydrochloric acid solution having a pH of 2.0 (2 grams ground, vacuumfreeze dried hide, 200 ml. dilute acid) for 15 minutes at roomtemperature, and then treated in a Waring Blendor for 25 minutes at atemperature not higher than 25 C.; about 16% of the solids was ofsubmicron size. The resulting gel had a pH of 3.0 and an apparentviscosity, measured at 25 C. on a HBT Brookfield viscosimeter with TBspindle, 10 rpm, of 42,900 centipoises.

Using an acid solution having a pH of 3.0, the product had a pH of 5.4,exhibited little bodying action (apparent viscosity under sameconditions 4800 cps.) and was replete with undispersed fibers. A stabledispersion was not formed even after prolonged mechanicaldisintegration. At a pH of 2.6, an apparent viscosity of about 30,000cps. was obtained. The viscosities of products rose to a peak when thepH of the treating acid solutions was in the pH range 2.0 to 2.3, anddropped off rapidly so that below a pH of 1.7, the viscosity was againbelow 30,000 cps. apparent viscosity, reflecting degradation of thecolla en and the formation of soluble lower molecular weight components.Using an aqueous hydrochloric acid solution having a pH of 1.3, a largeproportion of the hide substance is degraded to very low mo lecularweight material. The disintegrated product had a pH of 1.5 and aviscosity of less than cps.

The quantity of acid as set forth in this example is based upontreatment at a 1% solids concentration or consistency. As discussedhereinabove, when the concentration of collagen solids is increased,corresponding increases in the quantity of acid will be required toachieve the preferred final pH range of the dispersions.

The amount of submicron material may be measured by sedimentation, afterdilution of an aqueous gel to sufficiently low concentration so that theliquid is sufiiciently thin to allow heavy particles to separate out.Specifically, a 1% gel was prepared as above and diluted by at least afactor of ten. This was allowed to stand six hours, and the percentageof solids in the top fifth of the material was measured. This material,being in Brownian motion, is colloidal; moreover, inspection under themicroscope indicated it to be substantially all of submicron size. Thepercentage of submicron material in the total sample was calculated fromthe amount found in the top fifth. It is not essential that thisprocedure be followed precisely in measuring the content of submicronmaterial. So long as microscopic examination of the top aliquot revealsthe absence of substantially all material above 1 micron, the method maybe used.

EXAMPLE 2 TABLE 1 Viscesity, cps.

Visual observations Undispersed fibers. Illglbly viscous, transparentgel.

Do. Low viscosity, opaque gel.

Low viscosity, opaque gel presence of oil phase.

The foregoing examples illustrate the criticality of the pH of thetreating acid solution. These examples illustrate that where the initialpH of the acid solution exceeds the upper limit of about 2.6, the acidis insufiicient to eflect the three-fold action discussed hereinaboveand the attrited material contains a large quantity of fibers. Where theinitial pH of the acid is below the lower limit of about 1.6, thequantity of acid effects the three-fold action and in addition causes adegradation to low molecular weight acid-soluble products as evidencedby the extremely low viscosities of the dispersions.

The gels of this invention have one characteristic which differentiatesthem in kind from gels produced from tropocollagen gelatin: they exhibitonly a minor increase in viscosity immediately after making, and theviscosity then remains stable for days. Thus, the 1% gel of Example 1showed an apparent viscosity immediately after making of about 3,600centipoises (HBT Brookfield, spindle TA, 100 R.P.M., 25 C.); within anhour it increased to 4,100 centipoises; and it remained at essentiallythat viscosity over a storage period of six days. This is in sharpcontrast to the viscosity characteristics obtained with freely solubletropocollagen and its degradation products like gelatin; gels made fromthese materials show progressive and very major increases in viscosityand in body over similar periods.

Below 0.5% concentration, the aqueous dispersions are too thin to beconsidered true gels. A gel of the product of Example 1 is extremelythick, but it can be extruded through an orifice at high pressure. Gelscan be made with higher percentages of solids, but become difiicult tohandle with equipment other than high shear mixers such as Banburymills.

Gels made in accordance with this invention have another notablecharacteristic. Their apparent viscosity is markedly susceptible toshear rate; the 1% gel of Example 1 shows an apparent viscosity whichranges from about 400,000 centipoises at 0.5 R.P.M. to 3600 centipoisesat 100 R.P.M., in a straight line when plotted on logarithmiccoordinates. This reduction of apparent viscosity with increasing shearrenders the gels particularly useful as a bodying agent in water basepaints, to improve pigment suspension without loss of brushability.These gels exhibit striking non-Newtonian flow properties andthixotropic phenomena largely because of the dispersed rod-like natureof the constituent water-insoluble submicron microcrystalline colloidalparticles. Further, when the apparent intrinsic viscosities of thesegels are measured, values in excess of nine have been obtained,reflecting a remarkably high apparent molecular weight, as compared withtropocollagen having values of only 1 or 2 measured in the same manner.

Another unique and distinguishing characteristic of the products of thisinvention is their behavior when dried as compared to prior artproducts. As disclosed in US. Pat. No. 3,157,524, the drying of the acidgels prepared in accordance with that method produces a product whichwhen immersed in water reverts to a gel and is soluble in water. Thepatent teaches that in order to obtain a waterinsoluble product it isnecessary to neutralize the acid component of the gel and to remove thesalt formed by the neutralization reaction. Products obtained by dryingthe gels made in accordance with the present invention are insoluble inwater as described hereinbefore and as illustrated by the followingexample:

EXAMPLE 3 Twenty grams of chopped-up cowhide, free of water byfreeze-drying was placed in 1980 ml. of a hydrochloric acid solutionhaving a pH of 2 and treated at 25 C.- C. in a Cowles Dissolver, ModellVG, for 15 minutes at 5400 R.P.M., using a four-inch pick-blade. At theend of the attrition, the 1% gel of microcrystalline colloidal collagenwas spread in a freezedrying tray to form a layer Vs inch thick, andfreeze-dried overnight to C., vacuum five microns, heating cycle notexceeding 30 C. with condensation of sublimed water at C.). Theresultant product was a /8 inch mat which absorbed sixtyfive times itsown weight of water. The tensile strength of a dry test strip 1 inch inwidth was 3 /2 pounds, and the Wet strength of a like test strip wasquite low, but measurable.

Test samples of the mat can be repeatedly immersed in water, air driedand reimmersed in water without disintegrating. Upon immersing the matin water, it swells but retains its integrity even when allowed toremain in the water over periods of months. This action of the driedproduct illustrates that the original macromolecular morphology of themicrocrystalline colloidal particles has been retained to an extentsufficient to preclude disintegration to a true molecular dispersion oftropocollagen. In other words, the partial salt of collagen containssufiicient natural bonds that hold the original collagen moleculestogether in the collagen source material to render the productwater-insoluble in the presence of the bound acid.

The retention of a portion of the original bonds is also evident from aconsideration of the bound acid in the dry products. When the amino acidresidues of bovine corium collagen, for example, are considered, 1 gramof collagen contains approximately 0.78 millimole of primary aminogroups available to react with an added acid. Actual analyses ofproducts derived from microcrystalline colloidal collagen gels preparedwith various acids showed a bound acid content varying from about 0.4 toabout 0.7 millimole of acid (calculated as HCl) per gram of collagenwith an average bound acid content of about 0.58 millimole of acid pergram of collagen. Accordingly, it is concluded that certain of the aminogroups are bound in the inner region of the microcrystalline particlesand are unavailable to react with the acid.

As indicated above, other acids can be substituted for hydrochloric acidin the process described above, but concentrations of acids and rangesmust be varied. Moreover, when plotting gel viscosity obtainable againsthydrogen ion concentration in the acid solution, the peaks obtained withother acids may be sharper, indicating a much narrower usefulconcentration, indicating greater difiiculty in preventing degradationand build up of soluble tropocollagen. Sulfuric acid gives such a lowsharp peak that its use in commercial operations is counter-indicated;the peak is so narrow that control is much too difiicult. Acetic acid,hydrobromic acid and cyanoacetic acid all give high peaks, but muchnarrower peaks than hydrochloric acid, so that greater care must beexercised in their use; even when they produce high viscosity gels,variations in localized conversion to tropocollagen to make dry productswhich lose their integrity when immersed in water. From a practicalcommercial viewpoint and in view of its low cost and the absence ofcomplications in its use, hydrochloric acid is the treating agent ofchoice.

The optimum viscosity of the gels is obtained at different specific pHvalues of the gels when different acids are utilized as illustrated bythe following example:

EXAMPLE 4 Gels containing 1% solids were prepared from ground, vacuumfreeze-dried cowhide and acid solutions having different pH values asdescribed in Example 1. The initial pH of each solution and the pH ofeach final dispersion or gel were measured. The viscosities of thedispersions were determined as described in Example 1. The results ofthese determinations were as follows:

TABLE 2 Initial Viscosity, Acid pH Final pH cps.

RBI 3. 4. 60 4,000 2. 38 3. 67 44, 300 2.06 2. 57 28, 800 1. 35 1.42,200

H SO 3.02 4. 53 4, 000 2 4 2. 34 3. 45 25, 700 2.05 2. 55 19, 700 1. 50l. 60 6, 000

COOH 2. 98 3.51 39,400 CH3 2, 37 2. 58 28,000 2.06 2. 25 18,000 1. 42 1.58 1, 600

CNCH2CO0H 3. 00

It will be noted from the foregoing data that the specific maximumviscosity of the gels varies with the different acids. When these andother data are plotted using loglog co-ordinates, it is found that, ingeneral, maximum viscosities are obtained by the use of acid solutionshaving an initial pH of about 23:0.1 and when the pH of the gels isabout 32:0.2.

Obviously, a product which yields such highly viscous gels at lowconcentrations suggests its use for a very wide range of purposes. Asstated above, the properties of the gel render the product useful inwater base paints. As disclosed and claimed in copending applicationSer. No. 553,295, filed May 27, 1966, abandoned in favor of continuingapplication Ser. No. 32,437, filed Apr. 27, 1970, now Pat. No.3,649,347, dated Mar. 14, 1972, the product is particularly useful informing coatings on a wide variety of bases. The coatings may beprotective, decorative or may serve as a bonding agent, for example, asbetween fibers in a. non-woven fibrous web. The product is also usefulin foodstuffs, and cosmetic and pharmaceutical preparations which areingested or applied topically to humans and animals.

As disclosed and claimed in copending application Ser. No. 527,054,filed Feb. 14, 1966, now Pat. No. 3,471,598, dated Oct. 7, 1969, theproduct has utility for the production of freeze-dried mats or spongescharacterized by extremely high water and liquid absorbency renderingthe mats or sponges particularly useful as wound dressings, surgicalsponges and the like.

The freeze-dried mats are porous and find utility as filter elements.For example, a freeze-dried mat made from the dispersion of Example 1when used as a cigarette filter in comparison with conventionalcellulose acetate filters retained a greater proportion of the tobaccosmoke a determined by the color of the filters after smoking. Thefilters showed no shrinkage or other harmful effects from exposure tothe smoke; the cigarettes drew at least as freely as the cigaretteshaving conventional cellulose acetate filters.

In addition to the above utilities, aqueous dispersions can be extrudedto form water-insoluble fibers and films which can be used as is or thecollagen may be crosslinked by known techniques, as in tanning, to giveimproved products. The product can be dispersed with other fibers (e.g.,cellulose fibers) and made into paper where it serves as an innocuouswet strength binder; the resultant paper shows marked increase instrength, wet or dry, as compared with untreatedpaper. Moreover, theproducts act to disperse such paper-making fibers as viscose rayon,which are normally difiicult to disperse in a paper beater. Furthermore,as a thin coating on paper, the product provides a high gloss, flexiblefilm with greaseproof properties.

The products of this invention may be derived from a variety of collagensource materials and it is obvious that the raw material will beselected according to the desired end use of the product. Similarly, thespecific acid employed will be selected according to the end use of theproduct. For example, citric or lactic acids may be employed for edible,cosmetic and pharmaceutical uses whereas oxalic acid may be employed forindustrial uses. Because the product is derived from a naturallyoccurring protein source and no toxic ingredient need be used in thepreparation of the product, the product presents no problem of toxicitynor does it provoke allergies.

Microcrystalline colloidal collagen is particularly useful in a widerange of food products because of its properties. It is edible andnutritious, bland in both taste and odor, has very little, if any, colorand free of textural defects which could adversely affect the taste andmouth feel of the food products. Gels have a very smooth and pleasantmouth feel and when present in foodstuffs become an indistinguishablepart of the product.

Small proportions of microcrystalline colloidal collagen form highlyviscous thixotropic gels thereby allowing improvements in variousphysical properties of foodstuffs such as eating quality, appearance andtactual, visual and taste textures. Thus, for example, liquid foodstuffssuch as salad dressings may be thickened and insoluble ingredients maybe maintained in a relatively uniform dispersed state by incorporationof small proportions of the micro crystalline colloidal collagen. Thisform of collagen may replace gelatin to produce such products which willhave a substantially stable viscosity over a long period without agradual increase in viscosity and setting into a gummy mass as occurswith gelatin. The product is particularly well suited as an inexpensivereplacement for egg white and gelatin in various types of food productsbecause it produces the same effects at appreciably lowerconcentrations.

The various properties also render this new form of collagenparticularly satisfactory in a variety of cosmetic and pharmaceuticalcompositions or preparations. Certain pharmaceuticals and cosmeticcompositions include organic-soluble substances and are used in liquidform. As disclosed in copending application Ser. No. 499,077, filed Oct.20, 1965, now Pat. No. 3,393,080, dated July 16, 1968, stablemicrocrystalline colloidal collagen gels may be formed in aqueousliquids consisting essentially of water and up to about 65%, by weightof the liquid, of water-miscible organic solvents such as alcohols, forexample, methanol, ethanol, isopropanol and n-propanol; cyclic alcohols,for example, tetrahydrofurfuryl alcohol and furfuryl alcohol; ethers,for example, dioxane, tetrahydrofurane; and ketones, for example,acetone and methylethyl ketone. Accordingly, liquid or fiowable andpaste or ointment classes of compositions may be produced wherein acosmetic or pharmaceutical ingredient is dissolved in the liquid phasein which the microcrystalline colloidal collagen with or without otherinsoluble ingredients is dispersed.

Microcrystalline colloidal collagen is highly satisfactory in a widerange of pharmaceutical and cosmetic compositions or preparations, asstated above. It is particularly advantageous in liquid, paste and creamtype compositions because of the ability to form gels at lowconcentrations and the ability to maintain insoluble ingredients in astable dispersed state. It has a high affinity or sorptive power foroleaginous substances and will function as a means for dispersing theoleaginous substances in aqueous liquids. In lotion, emulsion, cream andointment types of compositions that conveniently include oily, fatty orwaxy (oleaginous) ingredients, the stability of the composition may beimproved by the addition of a small proportion of the colloidalcollagen. Microcrystalline colloidal collagen may be utilized to replacepart or all of the oleaginous ingredient. The ability to reduce oreliminate oleaginous ingredients in these types of compositions reducesthe greasiness of the products. Accordingly, after the composition isspread over the skin of the user and allowed to dry, the coatings have alower soiling tendency and the coatings are not readily transferred bycontact with clothing, bandages, etc.

This new form of collagen has excellent compressibility characteristicsand, when formed under compression, provides a coherent structure and,accordingly, finds value in the manufacture of tablets and compressedshapesrIt may be used to replace in whole or in part conventionalsubstances such as starch, sugar and binding agents such as gelatin,syrups and various gums. One of the characteristics of the tablets isthe dust free character as compared to conventional tablets. Also, inpowder composition, form of collagen may replace, in whole or in part,opacifying agents (clay, magnesia, zinc oxide, etc.), slip materials(talc, metal stearates, etc.), adherent materials (clay, stearates,etc.) and absorbents (chalk, kaolin, etc.).

The microcrystalline colloidal collagen is of particular advantage inthe preparation of hair sprays. It is well recognized that polyvinylpyrrolidone which is a common ingredient in commercial hair sprays isproving to be harmful because, in use, some of the hair spray is inhaledby the users. The polyvinyl pyrrolidone collects in the lymph glands andcannot be decomposed in the human body. The microcrystalline colloidalcollagen, on the other hand, is entirely harmless.

In shaving creams, soaps, bubble bath compositions, etc., this form ofcollagen has utility because of its ability to strengthen and stabilizethe lather or foam. It appears to function to strengthen the gasenclosing walls and prevent them from readily breaking down.

Although this form of collagen will form compositions with excellentadherence when applied to the skin of the user because of the excellentgel and dispersion forming characteristics, the coatings or films whichare formed are readily removed by the application of water. Althoughthis form of collagen is not soluble in water, it readily forms thedispersions and can be easily removed. Similarly, although the tabletswhich may be formed by compressing this form of collagen with the otheringredients of a pharmaceutical or cosmetic composition have excellentcohesive characteristics, the tablets readily disintegrate in water orin the stomach fluids. The rate of disintegration may be varied byvarying the pressures used in tableting and by the relative proportionsof the collagen and other ingredients.

The term pharmaceutical is intended to refer to drugs as defined in theUnited States Federal Food, Drug and Cosmetic Act. In view of itsinertness, microcrystalline colloidal collagen may be associated withlarge numbers of drugs, such as analgesics, anti-infectives, anti-acidpreparations, anti-ulcer drugs, anti-histamines, hypnotics, sedatives,vitamins, stomachics, astringents, fungicides, local antiseptics, etc.

The term cosmetic" is intended to include all types of products whichare applied in any manner directly to the person for the purpose ofcleansing or embellishment and is intended to also include toilet soaps,shaving soaps and creams as well as deodorants, depilatories, and suntanand EXAMPLE 5 Aspirin tablets were prepared having the followingcompositions:

Parts by weight of- Aspirin 85.0 85. 0 Corn starch 14. 5 14. 5Microcrystalline collagen 1% gel. 100 ll'lagncsinm stearatc 0. 5

In preparing the tablets, the aspirin was first milled with the cornstarch filler and subsequently wet mixed with a 1% gel ofmicrocrystalline collagen (ground hide collagen-citric acid). Afterforming the wet or damp mix, the mix was screened, dried andsubsequently dry screened. In forming the tablets identified as A, thedry screened material was blended with the magnesium stearate which wasused as a lubricant and the mixture then tableted in the conventionalmanner. In forming the tablets identified as B, after dry screening, thematerial was formed into tablets in a conventional manner. Both types ofmaterials had approximately the same flow properties and both forms oftablets showed substantially no dusting when placed in bottles andshaken.

EXAMPLE 6 A hair spray was formed having the following composition:

Parts by Weight In forming the mixture, the ingredients were added tothe alcohol and then the alcohol mixed with the water. Themicrocrystalline collagen (ground hide collagencitric acid) was thenadded and the mixture subjected to the necessary attrition. Forty partsof the mixture were then transferred to a standard aerosol can and 60parts of .the propellant,trichlorofluoromethane-dichlorodifiuoromethane, was added and thecontainer sealed. The spray when applied to hair provided what iscommonly termed a conditioning action, achieved desired hair bodyingeffects and possessed the desired holding properties.

EXAMPLE 7 A conventional cleansing cream, sample A, and an identicalcream containing microcrystalline colloidal collagen, sample B, wereprepared, the compositions of which were as follows:

Parts by weight of Beeswax 8. 4 8. 4 Mineral oil- 25. 0 25.0 Borax 0. 40. 4 Water 16. 2 16. 2 Microcrystalline collagen 0. 25

E13. Both creams had substantially the same lustrous pearly whiteappearance, although upon standing, water separated from cream A. CreamB remained unchanged. Both creams were easily movable over the skin.After removal from the skin by wiping with a facial tissue, cream Bappeared to leave the skin with a smoother feel.

EXAMPLE 8 A greaseless hand cream was prepared from the following:

Parts by Weight Isopropyl myristate 2.0 Isopropanol 8.0 Sodium cetylsulfate 1.0 Water 86.5 Glycerol 1.0 Microcrystalline collagen 1.5

This cream was very stable on storage. It rubbed in the hands quicklyand easily without soaping or giving a feeling of wetness. Moreover,after the application, the hands felt soft and essentially non-greasy.No impression was left on glassware, china, or metals when they werehandled subsequent to the application of the cream to the hands.

This cream was rich. It spread easily without dragging or slippingexcessively, and appeared to be slowly absorbed by the skin duringapplication. When applied to sore, chapped skin, it had a smoothingelfect and immediately relieved the feeling of dryness.

EXAMPLE 10 A hand lotion was made up comprising:

7 Parts by Weight Glycerine 40 Distilled water 47 Ethyl alcohol 10 Oilof peppermint 2 Sodium lauryl sulfate, q.s.

Microcrystalline collagen 1 The lotion was viscous but readily pourableand had a lustrous pearly white appearance. It rubbed smoothly over thehand, giving the impression of lubricity without greasiness; on dryingthe applied coating, no visible trace of microcrystalline collagen wasapparent.

EXAMPLE 11 A non-greasy type sunscreen type cream was prepared from thefollowing:

Parts by Weight Microcrystalline collagen 1.0 Cetyl alcohol 0.5 Stearicacid 5.0 Ethyl p-aminobenzoate 1.2 Isopropanol 5.0 Water 86.0 Glycerol1.3

The cream spread to a non-greasy, uniform, and occlusive film that gavegood protection against radiant energy.

14 EXAMPLE 12 A brushless shaving cream was prepared from the following:

Parts by Weight Microcrystalline collagen 1 Triethanolamine 1 Oleic acid2 Sodium lauryl sulfate, q.s.

Water 50 The ingredients were all thoroughly mixed together in a WaringBlendor to produce a cream which was placed in a jar and capped. It hada light, wet appearance resembling conventional brushless type shavingcream and had a pleasant odor. The preparation had a slippery, creamyfeel on the skin without being greasy. It was used as a brushlessshaving cream and retained its moist condition during the entire shavingperiod.

EXAMPLE 13 The following toothpaste was prepared:

The mixture was prepared in a Waring Blendor and was pack-aged inconventional tubes. An outstanding feature of this paste is that it didnot dry out when the tube was left without the cap.

EXAMPLE 14 An anti-perspirant was prepared containing:

Parts by Weight Stearic acid 15.5

Microcrystalline collagen 1.0 Sodium lauryl sulfate 1.5 Propylene glycol5.0 Water 54.0 Urea 5.0 Aluminum Sulfate 18.0

In formulating, two mixtures were prepared, one containingmicrocrystalline collagen and the stearic acid, and the other comprisingthe balance of the ingredients. Each mixture was heated to 70 C. andthen mixed. The resulting product was in the form of a paste that waseasily spreadable over the skin and had a non-greasy feel.

In the foregoing examples of cosmetic compositions, the microcrystallinecolloidal collagen was a powdered or finely ground hide which had beentreated with citric acid. In preparing the powdered material, hidecollagen was treated with a citric acid solution having .a pH of about2.3 and attriting the mixture in a Waring Blendor. The resulting gel wasfreeze dried and the freeze dried product finely ground to provide thepowdered material. Alternatively, where the mixing step in thepreparation of the cosmetic composition involves an attrition step, amixture of finely ground hide collagen and the required amount of citricacid may be used and the microcrystalline colloidal collagen is formedduring the attrition step.

In the cosmetic compositions, the cosmetic ingredient or ingredientsmust be compatible with microcrystalline colloidal collagen, is presentin the compositions in an available form and in an amount sufiicient soas to impart the characteristic cosemetic elfect. The microcrystallinecolloidal collagen in these compositions is indistinguishable butimparts the enhanced properties to the compositions as discussedhereinbefore.

15 As stated hereinbefore, during the acid treatment of the collagen,the ionizable acid reacts only with the available amino groups to form awater-insoluble salt of collagen. Some of the amino groups are withinthe crystallites and are hydrogen bonded to each other within thecollagen and thus are not accessible to the treating acid. The termwater insoluble, ionizable salt of collagen is used herein and in theclaims to designate this reaction product. The water-insoluble,ionizable salts of collagen are unique in their characteristic offorming aqueous gels containing /2% dispersed salt having a pH betweenabout 3.2:02, the gels having a substantially stable viscosity for atleast 100 hours at 5 C. when stored in a closed container. Normally, thenew physical form of the microcrystalline colloidal particles comprisingbundles of aggregated tropocollagen units will have diameters of atleast about 25 A. and lengths of from about that of the tropocollagenunit (3000 A.) to just under 1 micron. Colloidal particles having thesediameters but of lengths considerably less than that of thetropocollagen unit may be obtained by very severe and prolongedattrition whereby bundles become severed transversely.

Various changes and modifications may be made in .practicing theinvention without departing from the spirit and scope thereof and,therefore, the invention is not to be limited except as de fined in theappended claims.

I claim:

1. A composition of matter in the form of a cosmetic cream or lotioncomprising a mixture of at least one conventional cosmetic cream orlotion ingredient other than water and a stable dispersion in water of awaterinsoluble, ionizable, partial salt of collagen having a boundionizble acid content of from about 0.4 to about 0.7 millimole of acid-(calculated as HCl) per gram of collagen based upon collagen containingapproximately 0.78 millimole of primary amino groups per gram ofcollagen, being essentially free of tropocollagen and degradedderivatives thereof and being further characterized in that whencolloidally dispersed in water to form a /2% by weight dispersionwherein at least 10% by weight of the partial salt has a particle sizenot exceeding 1 micron, the dispersion exhibits a pH of about 32:0.2 andexhibits an essentially constant viscosity after about 1 hour of for atleast hours when stored in a closed container at 5 C., the cosmeticingredient being compatible with the partial salt of collagen, beingpresent in an available form and in an amount sufiicient to impart itscharacteristic cosmetic property to the composition.

2. A composition of matter as defined in claim 1 wherein the partialsalt of collagen is derived from bovine collagen.

3. A composition of matter as defined in claim 1 wherein the partialsalt of collagen is a hydrogen chloride salt of collagen.

4. A composition of matter as defined in claim 1 wherein the partialsalt of collagen is a citric acid salt of collagen.

' 5. A composition of matter as defined in claim 1 wherein thecomposition is in a pourable liquid form.

6. A composition of matter as defined in claim 1 wherein the compositionis in a gel form.

7. A composition of matter as defined in claim 1 wherein the compositionincludes an oleaginous material.

8. A composition of matter as defined in claim 1 wherein the compositionincludes glycerol.

References Cited UNITED STATES PATENTS 3,393,080 7/1968 Brdi et a1. l06-161 3,628,974 2/1970 'Battista 106125 3,691,281 9/1972 Battista 424SHEP K. ROSE, Primary Examiner U.S. Cl. X.R.

1. A COMPOSITION OF MATTER IN THE FORM OF A COSMETIC CREAM OR LOTIONCOMPRISING AMIXTURE OF AT LEAST ONE CONVENTIONAL COSMETIC CREAM ORLOTION INGREDIENT OTHER THAN WATER AND A STABLE DISPERSION IN WATER OF AWATER INSOLUBLE, IONIZABLE PARTIAL SALT OF COLLAGEN HAING A BOUNDIONIZABLE ACID CONTENT OF FROM ABOUT 0.4 TO HAVING A 0.7 MILLIMOLE OFACID (CALCULATED AS HCL) PER GRAM OF COLLAGEN BASED COLLAGEN CONTAININGAPPROXIMATELY 0.78 MILLIMOLE UPON COLLAGEN CONTAINING APPROXIMATELYLAGEN, BEING ESSENTIALLY FREE OF TROPOCOLLAGEN AND DEGRADED DERIVATIVESTHEREOF AND BEING FURTHER CHARACTERIZED IN THAT WHEN COLLODIALLYDISPERSED IN WATER TO FORM A 1/2% BY WEIGHT DISPERSION WHEREIN AT LEAST10% BY WEIGHT OF THE PARTIAL SALT HAS A PARTICLE SIZE NOT EXCEEDING 1MICRON, THE DISPERSION EXHIBITS A PH OF ABOUT 3.2+0.2 AND EXHIBITS ANESSENTIALLY CONSTANT VISCOSITY AFTER ABOUT 1 HOUR OF FOR AT LEAST 100HOURS WHEN STORED IN A CLOSED CONTAINER AT 5*C., THE COSMETIC INGREDIENTBEING COMPATIBLE WITH THE PARTIAL SALT OF COLLAGEN, BEING PRESENT IN ANAVAILABLE FORM AND IN AN AMOUNT SUFFICIENT TO IMPART ITS CHARACTERISTICCOSMETIC PROPERTY TO THE COMPOSITION.